Extraction using aqueous two-phase systems (ATPSs) is finding widespread application in separation, concentration, and purification of biological products such as proteins/enzymes, viruses, cells and other biomaterials from fermentation broth and cell culture media. Use of ATPE quite essentially reduces the water load, removal of cell debris, polysaccharides, nucleic acids etc. and reduces the number of subsequent processing steps. Major hindrances for the widespread adaptation of ATPE on industrial scale is the high cost of phase forming polymers and environmental problems arising due to the disposal of phase forming polymers after the extraction of biomolecules. Hence, there is a need for the polymer separation and recovery from ATPSs after extraction of biomolecules. Presently, polymer phase recycling is being employed after the extraction of biomolecules in order to improve the economics of the process. However, in this method there is chance for the contaminates which is previously present in the polymer phase getting into the phase system instead of separating from the biomolecules. This results in reduction in desired levels of purity of the biomolecules in large-scale operations. Conventional methods like evaporation, vacuum drying alone are economically unviable for the polymer recovery from ATPSs due to high-energy requirements and need of special equipments for the creation and maintenance of required vacuum. Also, these methods take longer duration for the complete recovery of PEG due to presence of high amount of water along with the polymer. Hence, there exists a need for an economically viable method for the complete recovery of PEG from spent two-phase systems after extraction of biomolecules. Additionally, a need exists for the application of external field such as microwave field coupled along with conventional hot air drying to separate and completely recover PEG from spent ATPS at an faster rate with minimal use of energy.
Reference may be made to (Harries and Yalpani, 1985, Polymer-ligands used in affinity partitioning and there synthesis. In “Partitioning of aqueous two-phase systems” Eds. Walter et al. pp 589-626, Academic Press, New York) recovery of PEG in dry form is accomplished by cooling to give recrystallization or by precipitation with ether. In this method use of chemicals such as ether is undesirable, even detrimental since the recovered polymer has to reused for the extraction of biomolecules. Moreover, additional processing steps are essential to remove ether. Also, another way to get dry PEG is by maintaining vacuum at a temperature of 110-120° C. In this method the recovery of PEG takes long time and the use of vacuum makes it economically unviable in large scale operations.
Reference is made to recycling of polymer-rich phase in two stage aqueous two-phase systems for the recovery of yeast bulk protein, pyuruvate kinase and fumerase (Palomares and Lyddiatt, 1996, J. Chrom. B, 680, 81-89). However, in this method of polymer is partially recycled along with the biomolecule and also protein purification level achieved is low when polymer is recycled due to presence of contaminants in the recycled polymer phase.
Reference is made to (Johnson, 1994, Methods in Enzymology, Vol. 228, pp-571) wherein copolymer of ethylene oxide and propylene oxide are used to form the phase systems. Those polymers are recovered by increasing the temperature. However, in this method the polymers were recovered as a mixture of both the polymers. It may be noted that all these methods referred here refer to recovery of polymers by precipitation, recrystallization or by vacuum or recycling of polymer back or recovery in the form of mixture of two polymers. These methods are employed for subsequent separation, extraction and purification of biomolecules by the formation of ATPSs. This method is more suitable for the recovery of only thermosetting polymer and is not suitable for the separation and recovery of PEG.
There are no reports available in the liter on the recovery of dry PEG from ATPSs with the application of external field such as microwave field.